DNA Repair Mechanisms of Self-Resistance to Genotoxic Secondary Metabolites

细菌、真菌和植物产生的次生代谢物通常具有抗菌性能，使这些化合物在农业、工业和医疗应用中具有重要意义。因此，重要的是要了解这些化合物如何发挥其毒性，以及产生这些化合物的生物体如何避免自身天然产物的毒性而自毁。该研究项目将通过以下方式造福社会：1)为各级受训人员提供密集的实验室定量结构生物学体验，包括博士后、研究生和本科生；2)为菲斯克大学的本科生提供暑期实践研究机会；3)为女性和代表性不足的群体提供就业机会；4)为所有受训人员提供教学和指导机会；5)让对STEM感兴趣的高中生接触生物医学研究；以及5)社区推广。范德比尔特大学文理学院和医学院首席调查员的任命提供了一个特别合作和包容各方的培训环境。本项目的重点是链霉菌的高毒性天然产物双卡霉素/CC-1065/yatakemyins家族，它们通过共价修饰DNA而显示出强大的抗菌和抗肿瘤特性。我们实验室和其他实验室最近的工作已经建立了DNA碱基切除修复(BER)作为这些和其他遗传毒性(DNA损伤)天然产物的自我抵抗机制。这些自抗性蛋白编码在生物合成的基因簇中，参与天然产物的合成和调节。虽然大多数关于生物合成基因簇的工作都集中在合成天然产物的酶上，但非合成蛋白质的作用，包括那些参与自身毒性抵抗的蛋白质，已经落后。该项目的长期目标是了解这些自我保护机制的作用，这些自我保护机制可以导致发现相关酶的新的生物和生化机制，并从重新设计的生产有机体中提高天然产物的产量。短期目标是了解链霉菌对多卡霉素/CC-1065/yatakemyins耐药的特殊误码率机制。将使用结构生物学、生物化学、遗传学和细胞生物学来表征每种化合物的毒性基础，以及催化这一重要的天然产品家族BER的前两步的独特DNA糖基酶和多嘌呤核酸内切酶的特异性和机制。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

DNA Repair Mechanisms of Self-Resistance to Genotoxic Secondary MetabolitesBacteria, fungi, and plants produce secondary metabolites that often have antimicrobial properties, making these compounds important in agricultural, industrial, and medical applications. It therefore is important to understand how these compounds exert their toxicity and how the organisms that produce them can avoid self-destruction from toxicity of their own natural products. This research program will benefit society by providing 1) intensive quantitative structural biology experience in the laboratory to trainees at all levels, including postdoctoral, graduate, and undergraduate, 2) hands-on summer research opportunities to undergraduates of Fisk University, a nearby historically black college/university, 3) employment opportunities to women and underrepresented groups, 4) teaching and mentoring opportunities to all trainees, 5) exposure of high school students interested in STEM to biomedical research, and 5) community outreach. The appointment of the Principal Investigator in the College of Arts and Science and the School of Medicine at Vanderbilt University provides an exceptional collaborative and inclusive training environment. This project focuses on the duocarmycin/CC-1065/yatakemycin family of highly toxic natural products from Streptomyces that exhibit potent antimicrobial and antitumor properties by covalently modifying DNA. Recent work from our lab and others has established DNA base excision repair (BER) as a self-resistance mechanism for these and other genotoxic (DNA damaging) natural products. These self-resistance proteins are encoded within the biosynthetic gene clusters involved in synthesis and regulation of the natural products. While most work on biosynthetic gene clusters has focused on the enzymes that synthesize the natural product, the roles of the non-synthesis proteins, including those involved in self-resistance to toxicity, have lagged behind. The long-term goals of this project are to understand the roles of these self-protection mechanisms, which can lead to discovery of novel biological and biochemical mechanisms of the enzymes involved, and to increased yield of the natural product from a re-engineered producing organism. The short-term goal is to understand the specialized BER mechanism of duocarmycin/CC-1065/yatakemycin resistance in Streptomyces. Structural biology, biochemistry, genetics, and cell biology will be used to characterize the basis for toxicity of each compound and the specificity and mechanisms of the unique DNA glycosylases and apurinic endonucleases that catalyze the first two steps in BER of this important family of natural products.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.